Glutamate is the predominant excitatory neurotransmitter in the brain;glutamate receptors participate in both healthy brain function and pathological conditions. The NMDA receptor (NMDAR) is a glutamate-gated ion channel that is highly permeable to calcium (Ca++), but can be blocked by magnesium (Mg++). Most NMDA receptors are tetramers of two NR1 and two NR2 subunits. Four NR2 subtypes are expressed in the brain (NR2A-D);subunit composition greatly affects NMDAR channel properties such as Mg++ block and Ca++ permeability. Controlled Ca++ influx is essential for normal cell function, but deviation from the tightly- regulated "healthy" amount of Ca++ influx is implicated in several diseases, including bipolar and anxiety disorders. NMDAR hypofunction is linked to schizophrenia, in which there is evidence for region-specific downregulation of NMDAR subtypes. Understanding the physiological implications of subtype-specific NMDAR properties will help reveal functional consequences of NMDAR regulation in mental illnesses. The objective of this proposal is to assess and identify structural determinants of NMDAR subtype- dependent physiological properties, which may suggest novel approaches to pharmaceutical treatment of mental illness. Recently, our lab found that several observed differences between NMDAR subtypes are primarily due to variation in Mg++ permeation and binding of permeant ions to an intracellular site. The following specific aims are proposed to further understanding of how NMDAR subtype-specific interactions with ions shape their functional properties: (1) Define NR2 subunit-dependent properties that are responsible for variations in NMDAR Mg++ block. Preliminary data suggest the exciting possibility that a single residue substitution underlies many NR2 subunit differences in channel properties, and that this residue is involved in intersubunit interactions. The hypothesis that interactions between NR1 and this single residue in NR2 subunits is responsible for variations among NMDAR subtypes in Mg++ block and permeation will be tested. (2) Explore the physiological and structural bases of NMDAR subtype-dependent variation in single-channel conductance and Ca++ permeation. Computational, electrophysiological, and molecular biological approaches, including computer modeling of NMDAR structure, site-directed mutagenesis, and recording current from whole cells and single channels, will be employed to explore physiologically meaningful differences between NMDAR subtypes. Results of this research will further understanding of the diversity of NMDAR properties. Because NR2 subtype regulation is altered in schizophrenia, knowledge in this area may lead to innovative treatments for this and other mental illnesses.